1. Field of the Invention
The present invention relates to the rapid, continuous flow, processing of tissue for microscopic examination, from fixation to impregnation.
2. Description of the Related Art
Conventional methods prepare tissues for histology by incubation in separate solutions of phosphate-buffered 10% formaldehyde for fixation, a series of increasing concentrations of ethanol for dehydration, and xylene for clearing tissue of dehydration agent, prior to impregnation. Because of the time required for this process, usually 8 hours or longer, it is customary to complete these separate stepsxe2x80x94fixation, dehydration, clearing, and impregnationxe2x80x94overnight in automated mechanical instruments designed for those tasks (see, for example, U.S. Pat. Nos. 3,892,197, 4,141,312, and 5,049, 510). A typical automated tissue processor (TISSUE-TEK) requires more than eight hours and is programmed to process batches of tissue samples as follows.
Such conventional methodology demands that the tissue specimens be sent from the operating room, medical office or other sites, to a pathology laboratory on one day; the tissue specimens be prepared overnight; and the pathologist render a diagnosis based on microscopic examination of tissue sections the next day at the earliest, almost 24 hours after delivery of the specimen to the laboratory (FIG. 1). In addition to the minimum one-day delay in giving a surgeon the benefit of a report from the pathologist, there are also problems associated with impeded work flow in the pathology laboratory necessitated by the requisite batch processing of specimens, the safety concerns that attend having instruments operating overnight, the risk of possible instrument failures and the need to monitor the instruments, and the waste of using large volumes of reagents for such processing when automated. Moreover, expensive measures are required to prevent exposure of laboratory personnel to fumes and toxic substances associated with the reagents used in this process. Also, the large volumes of solvent waste and paraffin debris produced by conventional methodology pollute the environment.
Conventional fixation and processing cause irreversible damage to the structure of DNA and particularly RNA that limits the application of genetic techniques for diagnosis and research. Consequently, most DNA and certainly RNA analysis require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques.
Histological diagnosis of a frozen section suffers from multiple disadvantages in comparison to sections prepared from paraffin blocks: the slide prepared from a frozen section xe2x80x9cdoes not possess . . . uniformity of qualityxe2x80x9d; xe2x80x9cit is technically more difficult for serial sections of the same specimen to be examinedxe2x80x9d; xe2x80x9cextreme caution must be exercised in cutting the specimen in order to ensure a sufficiently thin section and to avoid the possibility of damaging details of the specimenxe2x80x9d; and all the slides must be prepared xe2x80x9cwhile the tissue is in the initial frozen statexe2x80x9d because, xe2x80x9c[i]f the tissue is thawed and refrozen for sectioning, it is severely damagedxe2x80x9d (U.S. Pat. No. 3,961,097).
There is an ever present interest in expediting tissue processing and analysis for diagnostic purposes. Furthermore, recent healthcare focus has been directed to lessening the cost of various procedures including tissue processing. The costs of tissue processing are related to time, the space required for preparation and analysis, reagents (both the amount required for processing and handling discard), and the number of personnel required. More importantly, patients and their physicians depend on evaluation and diagnosis by the pathologist to guide treatment. Reducing the amount of time needed to complete tissue processing would lessen the anxiety experienced during the period between obtaining the specimen and delivering the pathologist""s report to the surgeon.
Others have recognized the need to shorten the time required for tissue processing, but they have made only modest improvements in the conventional methods. To accelerate tissue processing, U.S. Pat. Nos. 4,656,047, 4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No. 5,023,187 uses infrared energy. U.S. Pat. No. 5,104,640 disclosed a non-aqueous composition of a fixative, a stabilizing agent, and a solubilizing agent that adheres a blood smear to a slide. However, the aforementioned patents do not teach or suggest that the entire process of preparing diagnostic tissue slides could be accomplished in less than two hours, starting from fixation and ending with impregnation, with continuous throughput of samples. The present invention provides such a process.
It is an object of the invention to provide compositions for tissue processing and an apparatus and system for utilizing the same that reduces the time required for tissue processing and analysis, and reduces the cost thereof by reducing time, the size of the laboratory facility, the volumes of reagents used, and the number of personnel required. This allows conversion of existing practice to rapid response surgical pathology for the patient undergoing an operation, and may even allow point-of-care diagnosis by the pathologist in the vicinity of the operating room.
With regard to the processing and analysis of solid tissue, a tissue slice must be on the order of 4 to 6 microns to be examined under a microscope, whereas the thinnest slice of fresh tissue that can be obtained by cutting is about 1 mm with the typical slice being on the order of 3 mm. In order to produce a sufficiently thin slice from microscopic examination, it is necessary to harden the tissue so that a finer slice can be obtained, e.g., by sectioning with a microtome. The present invention greatly accelerates the tissue hardening process and thus turns the conventional overnight processing into a process which totals on the order of 40 minutes. Thus, we have developed a simple, safe, low cost, expeditious, and reliable method that permits preparation of impregnated tissue blocks suitable for microtome sectioning in less than two hours from the moment tissue is received in the pathology laboratory. This method allows continuous flow of specimens, is adaptable to automation, precludes the need for formalin and xylene with their noxious fumes, allows standardization of tissue processing, and requires considerably smaller volumes of reagents than conventional methods. Either fresh or previously fixed tissues can be processed by the present invention.
In addition to the reduction in time required for tissue processing, the rapid preparation of tissue by the present invention is capable of preserving tissue structures and morphology that were lost with conventional methods.
Moreover, studies with tissues processed with the invention disclosed herein indicate better preservation of DNA and particularly RNA extraction than with conventional processing methods. Thus, tissues obtained in hospitals and other settings can be processed for both histologic and genetic studies soon after delivery to the laboratory, and archival material may be made available for future research and other applications. Improvements may be expected in the yield of genetic material, the stability of the genetic material in archival form, the size and integrity of the genetic material, and reducing chemical modification of the genetic material in comparison to the prior art.
An object of the invention is to provide a method and an apparatus for rapid processing of tissue for histology with continuous throughput. By xe2x80x9ccontinuous throughput,xe2x80x9d we mean accessing the system with additional samples, minutes apart. Therefore, at any given time there are samples of tissue in different stages of processing. In other words, with our method, there is continuous throughput and flow of specimens along the various steps of tissue processing. In contrast with our method, batch processing is presently required because conventional methodology takes eight hours or longer. Samples are placed in automated instruments, which can not be access with additional samples until the entire instrument cycle is completed. All these tissue samples are at the same stage of processing at any given step of the instrument cycle.
Yet another object of the invention is to provide non-aqueous reagents for rapid, continuous flow processing of tissue for histology.
A further object of the invention is to eliminate the need for toxic substances such as formalin and xylene in tissue processing.
In accordance with one aspect of the invention, a tissue specimen is fixed, dehydrated, and fat is removed. A suitable admixture for use is a non-aqueous solution comprised of fixative and dehydrating agents, preferably a ketone and an alcohol; the volume ratio of alcohol to ketone may be between about 1:1 to about 3:1. The tissue specimen is incubated for about 25 minutes or less, more preferably for about 15 minutes or less, and even more preferably for about 5 minutes or less. Incubation is preferably between about 30xc2x0 C. and 65xc2x0 C., more preferably between about 40xc2x0 C. and 55xc2x0 C., and most preferably between about 45xc2x0 C. and 50xc2x0 C.
Another aspect of the invention is fixation, dehydration, fat removal, and clearing of a tissue specimen. A preferred solution in this aspect of the invention is alcohol and a clearant. This process may be accomplished in about 5 minutes or less.
In yet another aspect of the invention, a tissue specimen is cleared and impregnated in a single solution comprised of a clearant and an impregnating agent. Preferably, this process may be accomplished in about 5 minutes or less. Prior to sectioning, the impregnated tissue specimen may be embedded in the impregnating agent.
A tissue specimen which has been fixed, dehydrated, and defatted may then be impregnated in a wax solution. Consistent with dehydration of the tissue specimen, the wax solution is preferably as low as possible in water content. Thus, the wax solution may be prepared prior to impregnation by heating the wax to evaporate any dissolved water and by degassing under reduced pressure. Impregnation of the tissue specimen may take place under less than atmospheric pressure and at elevated temperature to remove any solvents from the tissue specimen and to draw the wax solution into the tissue specimen. Vacuum decreases impregnation time by accelerating diffusion and reducing the evaporation temperature of any solvents that may be present in the sample. The wax solution may comprise degassed paraffin and/or mineral oil. Impregnation of the tissue specimen may be completed in about 15 minutes or less; preferably, completed in about 10 minutes or less. Prior to sectioning, the impregnated tissue specimen may be embedded in the impregnating agent to form a tissue block.
Another embodiment of the invention is processing a tissue specimen from fixation to impregnation in a series of solutions, at least some of which are admixtures that perform more than one task at the same time: fixation, dehydration, removal of fat, and impregnation. The admixture may include a fixative, a dehydrating agent, and a fat solvent (e.g., ketone and alcohol). Another solution may include fixative, dehydrating agent, fat solvent, and clearant (e.g., alcohol and xylene). Yet another solution may include a clearant and an impregnating agent (e.g., xylene and paraffin). The tissue specimen may be impregnated in a wax solution comprised of a mixture of different chain lengths (e.g., at room temperature, mineral oil which is liquid and paraffin which is solid). Preferably, an admixture contains at least two different chemicals (e.g., two alcohols).
Processing time may be reduced by a non-aqueous admixture (e.g., fixative-dehydrating agent-fat solvent, fixative-dehydrating agent-fat solvent-clearant, clearant-impregnating agent), microwave energy as a source to achieve uniform heating within the tissue specimen, and reducing the pressure by using a vacuum source. Diffusion of the solution into the tissue specimen and chemical exchange may be promoted by mechanical agitation, heat, reduced pressure, or a combination thereof.
The above steps may be accelerated by adding a fixative enhancer, a surfactant, or both to the solutions used in the process. The fixative enhancer may be polyethylene glycol (PEG), mono- and dimethyleneglycol, propylene glycol, polyvinyl pyrrolidone, or the like; the polymer used may be between about 100 and about 500 average molecular weight, preferably about 300 molecular weight. The surfactant may be dimethyl sulfoxide (DMSO), polyoxyethylene sorbitan esters (e.g., TWEEN 80), sodium dimethyl sulfosuccinate, mild household detergents, or the like.
The fixative may be a ketone (e.g., acetone, methyl ethyl ketone), aldehyde (e.g., acetylaldehyde, formaldehyde, glutaraldehyde, glyoxal), alcohol (e.g., methanol, ethanol, isopropanol), acetic acid, lead acetates and citrate, mercuric salts, chromic acid and its salts, picric acid, osmium tetroxide, or the like.
The tissue specimen may be dehydrated with methyl alcohol, isopropyl alcohol, ethyl alcohol, propyl alcohol, butanol, isobutanol, ethyl butanol, dioxane, ethylene glycol, acetone, amyl alcohol, or the like.
Fat may be removed from the tissue specimen with an organic solvent such as, for example, acetone, chloroform or xylene.
The clearant may be xylene, limonene, benzene, toluene, chloroform, petroleum ether, carbon bisulfide, carbon tetrachloride, dioxane, clove oil, cedar oil, or the like.
The tissue specimen may be impregnated and/or embedded in paraffin, mineral oil, non-water soluble waxes, celloidin, polyethylene glycols, polyvinyl alcohol, agar, gelatin, nitrocelluloses, methacrylate resins, epoxy resins, other plastic media, or the like.
In the context of the invention, a xe2x80x9ctissue specimenxe2x80x9d is a piece of tissue that may be processed by the methods disclosed herein. It may also refer to single cells from any biological fluid (e.g., ascites, blood, pleural exudate), or cell suspensions obtained from aspiration of solid organs or lavage of body cavities. Single cells may be pelleted by sedimentation or buoyant centrifugation prior to processing.
The methods of the invention are specially suitable for tissue specimens in which cell-cell contact, tissue organization, organ structure, or a combination thereof must be preserved. Such a specimen is a tissue slice preferably about 3 mm or less in its smallest dimension, more preferably about 2 mm or less, even more preferably about 1.5 mm or less, and most preferably about 1 mm or less.
The tissue specimen may be fresh, partially fixed (e.g., fixation in 10% forrnalin for 2-3 hours), or fixed (e.g., overnight fixation in 10% formalin or any other fixative). The above invention allows processing of a tissue specimen from fixation to impregnation in less than about two hours, preferably less than about 90 minutes, more preferably less than about one hour, even more preferably less than about 45 minutes, and most preferably less than about 30 minutes. If the tissue specimen is fixed or partially fixed, then the processing time may be shortened accordingly. Tissue may be transported from the operating room to the pathology laboratory in an aqueous solution; such a transport solution may consist of equal volumes of an aqueous buffer and the non-aqueous admixture described herein.
Following impregnation, the tissue specimen can be embedded to produce a block. The agent used to embed the tissue specimen is preferably the same as the material used for impregnation, but a different impregnating agent may also be used. The blocked tissue specimen can be mounted on a microtome to produce tissue sections of between about 1 micron and about 50 microns, preferably between about 2 microns and about 10 microns. The tissue sections may be further processed for histochemical staining, antibody binding, in situ nucleic acid hybridization/amplification, or a combination thereof. The tissue specimens are then typically examined by microscopy, but other techniques for detecting cellular properties may be used to examine the processed tissue specimen (e.g., automated cytometry, autoradiography, electrophoresis of nucleic acid).